FIH-1 Disrupts an LRRK1/EGFR Complex to Positively Regulate Keratinocyte Migration

Factor inhibiting hypoxia-inducible factor 1 (FIH-1; official symbol HIF1AN) is a hydroxylase that negatively regulates hypoxia-inducible factor 1α but also targets other ankyrin repeat domain–containing proteins such as Notch receptor to limit epithelial differentiation. We show that FIH-1 null mutant mice exhibit delayed wound healing. Importantly, in vitro scratch wound assays demonstrate that the positive role of FIH-1 in migration is independent of Notch signaling, suggesting that this hydroxylase targets another ankyrin repeat domain–containing protein to positively regulate motogenic signaling pathways. Accordingly, FIH-1 increases epidermal growth factor receptor (EGFR) signaling, which in turn enhances keratinocyte migration via mitogen-activated protein kinase pathway, leading to extracellular signal–regulated kinase 1/2 activation. Our studies identify leucine-rich repeat kinase 1 (LRRK1), a key regulator of the EGFR endosomal trafficking and signaling, as an FIH-1 binding partner. Such an interaction prevents the formation of an EGFR/LRRK1 complex, necessary for proper EGFR turnover. The identification of LRRK1 as a novel target for FIH-1 provides new insight into how FIH-1 functions as a positive regulator of epithelial migration.CME Accreditation Statement: This activity (“ASIP 2014 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“ASIP 2014 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.The asparaginyl hydroxylase factor-inhibiting hypoxia-inducible factor 1α (FIH-1; official symbol HIF1AN) was originally identified as a protein that interacts with and inhibits the activity of hypoxia-inducible factor 1α (HIF-1α) in the C-terminal transactivation domain^1,2 by coupling the oxidative decarboxylation of 2-oxoglutarate to the hydroxylation of HIF-1α.³ Significantly, proteins containing the ankyrin repeat domain, such as Notch, are other substrates for FIH-1.³ Only recently has FIH-1 been recognized to have pleiotropic roles in maintaining epithelial homeostasis.^4,5 For example, FIH-1 negatively regulates glycogen metabolism in corneal epithelium in a HIF-1α–independent manner via the direct involvement of the Akt/glycogen synthase kinase 3β signaling pathway.⁴ Furthermore, in epidermal and corneal epithelial keratinocytes, FIH-1 was shown to act as a negative regulator of differentiation via a coordinate decrease in Notch signaling.⁵ What is not clear in these studies is whether FIH-1 affects other signaling pathways known to influence keratinocyte growth, differentiation, and migration. For example, the regulation of Notch 1 activity by FIH-1⁵ raises the possibility of cross-talk with the epidermal growth factor receptor (EGFR)-signaling pathway, since EGFR signaling has been shown to be a negative regulator of Notch 1 gene expression and activity in keratinocytes.⁶Once EGF binds to the EGFR, numerous signaling pathways are activated that impact on cell proliferation, migration, differentiation, and survival.^7–9 With respect to the skin, EGFR impacts on epidermal and hair follicle development, keratinocyte proliferation, survival, cancer, and immune homeostasis.¹⁰ EGFR signaling also plays a prominent role in epidermal and corneal epithelial migration and wound repair. For example, in the epidermis, EGFR signaling has been shown to promote keratinocyte migration and wound repair.¹¹ Likewise, corneal perturbations activate the EGFR and downstream Ras-Raf-Mek-Erk1/2 (Ras, Raf, mitogen-activated protein kinase kinase, extracellular signal–regulated kinase 1/2) and phosphoinositide 3 kinase–Akt signaling cascades, which are required for efficient wound healing and are attenuated in patients with diabetic keratopathies.^12–14The activation of EGFR also commences endocytic trafficking, whereby the receptor is either packaged in lysosomes for degradation or recycled to the cell surface.^15,16 Endosomal trafficking is essential for establishing the extensiveness of the EGF-mediated signal, and thus much attention has been directed toward understanding the steps involved in the movement of the EGFR from the cell surface to cytoplasmic vesicles, such as the endosome, multivesicular body, and lysosome.^16–18 Recently, leucine-rich repeat kinase 1 (LRRK1) was recognized as a key regulator of EGFR endosomal trafficking.^19,20 Specifically, it is believed that LRRK1 forms a complex with activated EGFR through an interaction with growth factor receptor–bound protein 2 and that this complex is internalized in early endosomes.¹⁹ The mechanism by which LRRK1 regulates EGFR transport is from early to late endosomes.¹⁹LRRK1 protein kinase is one of the ROCO proteins, which contain a GTPase-like domain [Ras of complex proteins (Roc)] and a C-terminal of Roc (COR) domain.²¹ ROCO proteins have a series of leucine-rich repeats and/or ankyrin repeats, with LRRK1 containing six N-terminal ankyrin repeats.²² This latter aspect of LRRK1 is noteworthy since, as mentioned above, proteins with ankyrin repeat domains are potential substrates for FIH-1.³ Thus, FIH-1 has the potential to directly interact with LRRK1, which could impact on EGFR signaling.Here we show that ectopic expression of FIH-1 in keratinocytes increases phosphorylation of the EGFR, which positively affects keratinocyte migration via stimulation of the mitogen-activated protein kinase pathway, resulting in an increase in phosphorylated ERK1/2. Such enhanced migration is independent of Notch signaling. Moreover, our studies reveal that FIH-1 interacts with LRRK1 and prevents the formation of an EGFR/LRRK1 complex necessary for proper EGFR turnover.¹⁹ The identification of LRRK1 as a substrate for FIH-1 provides new insight into how FIH-1 functions as a positive regulator of epithelial migration. Thus, the breadth of FIH-1 epithelial biology is considerably larger than previously realized.     

The effect of healing phenotype-inducing cytokine formulations within soft hydrogels on encapsulated monocytes and incoming immune cells

The adverse immune responses to implantable biomedical devices is a general problem with important consequences for the functionality of implants. Immunomodulatory soft hydrogel-based interfaces between the implant and the host can attenuate these reactions. Moreover, encapsulation of the patient''s own immune cells into these interfaces can lead to the personalisation of implants from the immune reaction point of view. Herein, we described a co-crosslinkable composite hydrogel (composed of gelatin and hyaluronic acid), which could be used for the encapsulation of macrophages in the presence of an anti-inflammatory phenotype-fixing cytokine cocktail. To mimick the incoming immune cells on the coating surface in vivo, peripheral blood mononuclear cells were seeded on the hydrogels. The encapsulation of monocytic cells into the composite hydrogels in the presence of cytokine cocktails at 5× or 10× concentrations led to the spreading of the encapsulated cells instead of the formation of clusters. Moreover, the secretion of the anti-inflammatory cytokines IL-1RA and CCL-18 was significantly increased. The attachment of PBMC to the surface of the hydrogel is dependent on the hydrogel composition and also significantly increased in the presence of the cytokine cocktail together with the number of CD68+ cells on the hydrogel surface. Our study demonstrates that the delivery of a polarisation cocktail with biocompatible hydrogels can control the initial response by the incoming immune cells. This effect can be improved by the encapsulation of autologous monocytes that are also polarised by the cytokine cocktail and secrete additional anti-inflammatory cytokines. This interface can fine tune the initial immune response to an implanted biomaterial in a personalised manner.

Hydrogels made from the derivatives of gelatin and hyaluronic acid were used as coatings to control the immune responses.     

Spontaneous Pneumothorax in Children with Osteosarcoma: Report of Three Cases and Review of the Literature

Spontaneous pneumothorax is a rare manifestation of primary lung cancer or metastasis. It is estimated that < 1% of all cases of spontaneous pneumothorax are tumor-associated and metastatic osteogenic or soft-tissue sarcomas are associated most commonly with pneumothorax especially in the setting of cytotoxic chemotherapy or radiotherapy. In this article, we report three pediatric cases with osteosarcoma that developed spontaneous pneumothorax during chemotherapy with a review of the literature. Two of them had lung metastasis at the time of the detection of pneumothorax and the remaining patient was found to have a bronchopleural fistula.

SPx is an emergency situation and early diagnosis and management can improve prognosis and quality of life of the patient however the optimal management has yet to be determined.     

Body Composition Analysis of Patients on Waiting List for Cadaveric Renal Transplantation: A Comparison of Hemodialysis and Peritoneal Dialysis Patients

BackgroundBioelectrical impedance analysis is a simple, noninvasive method of assessing body composition. Dialysis modality and selection of buffer type may have an impact on body composition. The aim of our study was to compare body compositions of patients from the waiting list for cadaveric renal transplantation according to the dialysis modality.MethodsWe examined a total of 152 (110 hemodialysis [HD] and 42 continuous ambulatory peritoneal dialysis [CAPD]) patients. Demographic data were collected from patient charts. The last 6 months routine laboratory evaluations including hemoglobin, serum creatinine, intact parathyroid hormone, albumin, C reactive protein, calcium, phosphorus were collected. Body compositions were measured using the Tanita BC-420MA Body Composition Analyzer (Tanita, Tokyo, Japan). We made a subanalysis of the CAPD group according to buffer choices as follows: lactate-buffered (n = 16) and bicarbonate/lactate–buffered (n = 26) solution users.ResultsThe body weight (P = .022), body mass index (BMI; 25.8 ± 4.7 vs 23.4 ± 4.9 kg/m², P = .009), muscle mass (P = .01), fat-free mass (P = .013), and visceral fat ratio (9.5 ± 5.4 vs 7.3 ± 4.1 %, P = .022) were significantly higher in the CAPD group. Total body water of CAPD patients were also higher (P = .003), but total body water ratios of HD and CAPD groups were similar. Fat and fat-free mass ratios of patient groups were also similar. Comparing CAPD subgroups we observed that patients using bicarbonate/lactate–buffered solutions had higher body weights (P = .038), BMI (27.1 ± 5 vs 23.7 ± 3.5 kg/m², P = .018) values, and visceral fat ratios (8.0 ± 5.2 vs 4.6 ± 2.5 %, P = .023). These patients also tend to have higher fat mass without statistical significance (P = .074). Fat, muscle, and fat-free mass total body water ratios of peritoneal dialysis subgroups were similar.ConclusionWe believe that body composition analysis should be used as a complementary method for assessing nutritional status of PD and CAPD patients as body weight or BMI measurements do not reflect fat, muscle masses, and visceral fat ratios in these patients. Stable, well nourished CAPD patients should be closely observed and be encouraged to increase daily exercise and/or decrease calorie intake from other sources to decrease risks associated with abdominal obesity.     

Dialysis Patients With the Metabolic Syndrome Need Less Recombinant Human Erythropoietin for Similar Hemoglobin Levels

IntroductionThe metabolic syndrome (MS) components, such as dyslipidemia, prothrombotic status, and increased blood pressure, are risk factors for patients with renal disease. Visceral fat mass is closely related to the MS and atherosclerosis. We investigated the effects of body compositions and MS on anemia parameters and recombinant human erythropoietin (rHuEPO) requirements in maintenance hemodialysis patients.MethodsBody composition (body mass index and bioimpedance analysis) and laboratory data were obtained from 110 dialysis patients. The MS was identified according to ATP-III criteria. Anemia parameters, hemoglobin (Hgb), albumin, C-reactive protein (CRP), calcium, phosphorus, parathormone levels, and rHuEPO requirements over the last 6 months were retrospectively analyzed.ResultsPatients with the MS seem to reach target Hgb levels more frequently (10–12 g/dL; 66.3% vs 84.8%; P = .03) without any difference in total intravenous iron therapy dosage. MS patients also required lower rHuEPO for reaching similar Hgb levels compared with patients without MS (2679.3 ± 1936.1 vs 3702.5 ± 2213.0 U/kg/6 mo; P = .02). There were no differences in serum CRP, albumin, or Hgb levels between the 2 groups (P > .05). We observed that patients with MS had significantly higher fat mass and visceral fat ratio, but similar muscle mass values compared with no-MS counterparts (P = .0001 and .001, respectively). However, when we compared the ratios of these parameters we observed a significant reduction in muscle ratios and a significant increase in fat ratios of MS patients (P = .0001).ConclusionOur results indicate that MS might be an advantage for reaching higher Hgb levels with lower rHuEPO dosages. The possible reason for this might be the good nutritional state and increased fat mass of patients with MS.     

Low plasma renin activity and high aldosterone/renin ratio are associated with untreated isolated systolic hypertension

Abstract Objective. Isolated systolic hypertension (ISH) is generally encountered in elderly patients and there are scarce data regarding the renin-angiotensin-aldosterone system (RAAS) activity in patients with ISH. We aimed to determine the plasma renin activity (PRA), plasma aldosterone levels (PAL) and aldosterone/PRA ratio (PAL/PRA) in patients (age >50 years) with ISH and to compare these values with patients with essential hypertension (EH) as well as subjects with normal blood pressure values (control) who have similar age and cardiovascular risk profile. Methods. Consecutively, 42 untreated ISH patients, 30 patients with EH and 29 normal subjects were included in the study. Parameters were presented as median (interquartile range). Results. There were no significant differences regarding age, gender and other cardiovascular risk factors among groups. As expected, systolic, diastolic blood pressure and pulse pressure values were significantly different among groups. Besides, PRA values were found to be significantly lower in patients with ISH (0.4 [0.2-1.1] ng/ml/h) compared with the EH (0.95 [0.5-2.6] ng/ml/h, p =0.024) and control (1.3 [0.7-2.1] ng/ml/h, p =0.001) groups. Although, PAL were similar among groups, PAL/PRA ratio was significantly higher in ISH group (134.1 [73-224]) compared with those with EH (42.2 [35-84], p <0.001) and the control group (53.3 [30-106], p =0.001). No significant difference was present with respect to PAL/PRA ratio between EH and control groups. Conclusions. Our findings suggested that in patients with ISH, despite lower PRA levels, PAL/PRA ratio is significantly higher compared with the patients with EH and subjects with normal blood pressure. Since higher PAL/PRA levels is an indicator of relative aldosterone excess, medications blocking RAAS activity including aldosterone antagonists may have useful cardiovascular consequences in addition to their antihypertensive effects in ISH.     

Paraoxonase Activity in Glomerulonephritic Patients

Background. Cardiovascular disease is the most common cause of morbidity and mortality in patients with chronic renal failure. Glomerulonephritic patients have an increased risk for cardiovascular disease, but its etiology is unclear. It is known that an increase in oxidizability of apolipoprotein B-containing lipoproteins has a key role in the initiation of atherosclerosis, and paraoxonase enzyme activity particularly has a preventive role against atherosclerosis. The aim of the present study was to evaluate the oxidizability of apolipoprotein B-containing lipoproteins, serum, and urinary paraoxonase/arylesterase activities in glomerulonephritis patients who had normal lipid parameters and creatinine levels. Methods. Thirty-two patients with glomerulonephritis and 22 healthy controls were included in this study. A total of 32 patients (including nine with membranous GN, eight with immunoglobulin A nephropathy, eight with mesangial proliferative GN, five with focal-segmental glomerulosclerosis, one with diffuse proliferative GN, and one with minimal chance disease having biopsy proven GN) were enrolled into the study. We compared serum and urinary paraoxonase, arylesterase, serum lipids, urea, creatinine, hemoglobin, total protein and albumin values between groups. Results. Serum urea, creatinine, total protein, albumin, uric acid, hemoglobin, and lipid parameters were similar in the glomerulonephritis and control groups (p > 0.05). PON1 activity was significantly lower in GN group than controls, but there was no statistically significant difference on arylesterase activity between groups. Oxidizability of apolipoprotein B-containing lipoproteins was significantly higher in GN group than controls. Conclusion. Our study shows that the findings of normal serum levels of creatinine, lipids, and proteins increased the oxidizability of apolipoprotein B-containing lipoproteins, and any decrease in PON1 activity in patients diagnosed with GN should be considered important. Hence, the immediate commencement of preventive as well as curative treatment in other to avoid the risk of cardiovascular and renal problems would be a correct approach.     

Computerized analysis of occlusal contacts in bruxism patients treated with occlusal splint therapy

PURPOSE

Occlusal splints are commonly used to prevent tooth wear caused by bruxism. However, the effects of splints on occlusion are still unclear. Although it is rarely alluded in literature, splints can provoke severe occlusal alterations and other complications. This study was aimed to identify differences in the responses of individuals with bruxism and healthy individuals to a full-arch maxillary stabilization splint in terms of occlusal changes.

MATERIALS AND METHODS

Occlusal contacts in 20 (5 male, 15 female) bruxism patients and 20 (5 male, 15 female) controls with normal occlusion were evaluated before and after occlusal splint therapy. T-Scan III, a computerized occlusal analysis system, was used to simultaneously measure occlusion and disclusion times as well as left-right and anterior-posterior contact distributions before splint therapy and 3 months after therapy. Wilcoxon and Mann-Whitney U tests were used for statistical analyses (α=.05).

RESULTS

No differences were found in the posterior contact of bruxism patients before and after stabilization splint treatment. However, differences in posterior contact were observed between bruxists and normal individuals prior to treatment, and this difference disappeared following treatment.

CONCLUSION

The results of this study showed the use of a stabilization splint may not have an effect on occlusion. However, the area of posterior occlusal contact among bruxists was found to be greater than that of normal individuals. According to this study, the clinical use of splints may be harmless.